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Revision as of 19:56, 20 August 2012
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== Lab 9: Series 3
Reverse Genetics
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Induction of HT115(DE3) feeding strains for RNAi
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== Lab 9: Series 3
Investigating Gene Regulation Using RNAi
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Through reverse genetics we will deduce the function of a gene starting with its sequence and working back to its phenotype. There are many genes in the genome whose phenotype when mutated is lethal; therefore, it's impossible (or very difficult) to tie function to a particular gene in the traditional forward genetics manner of creating random mutations, looking for phenotype changes, and then finding the defective gene responsible
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that function.
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Placeholder
for
Future information
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In our reverse genetics study of some interesting ''C. elegans'' genes, two different strains of worms, wild-type and rrf-3 (RNAi enhanced), are fed bacteria expressing dsRNA specific to a particular worm gene. Ingesting dsRNA initiates cascade of events that leads to the destruction of the mRNA of the target gene. An altered phenotype in the progeny of RNAi-treated worms indicates what happens when the normal function of this gene is lost or significantly downregulated.
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Double stranded RNA (dsRNA) can be introduced to the ''C. elegans'' cells in many different ways including: feeding, injection and soaking. Each of these methods has positives and negatives. We are using the feeding method - where we use genetically modified bacteria as dsRNA factories.
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To begin to investigate the power of reverse genetics, you will need to grow your own '''induced''' bacteria to seed your plates for RNAi feeding.
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'''Done on the night before this lab:'''
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You and your partner made an overnight broth culture of your selected colony. This process created a sub-culture of many identical copies of the plasmid carrying the construct that will induce RNAi to silence or downregulate the gene that you want to study.
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'''On the morning of lab:'''
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Your instructor or the lab staff will come in early in the morning and '''sub-culture''' your bacterial overnight. The cells will be in stationary phase in the morning and successful induction requires log phase growth.
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To create the subculture of bacteria your cultures will be diluted 1:5 (1 mL of culture into 4 ml of LB + amp). These cultures will be allowed to grow until lab time - approximately 3-4 hours.
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When you come in to lab you will induce your cultures to make lots of dsRNA by adding IPTG to the culture and letting it continue to incubate for a few hours so the cell is full of dsRNA. The IPTG will compete with the repressors on the lac o promoter and remove them and allow the gene for T7 RNA polymerase to be transcribed and then translated into the RNA polymerase protein. The T7 RNA polymerase then binds to the T7 promoters on the pL4440 plasmid and transcribes our ''C. elegans'' DNA into RNA!
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A simplified map of the ''C. elegans'' RNAi plasmid :
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[[Image:L4440.tif]]
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More specifically: The bacteria cells of strain HTll5(DE3) contain the T7 RNA polymerase gene (contained within a stable insertion of a modified lambda prophage λ DE3) under the control of lac operon regulatory elements. This allows expression of T7 polymerase to be controlled by isopropyl-β-D-thiogalactopyranoside (IPTG), a lactose analogue that induces expression of genes under the control of the lac operon o gene. When IPTG is added, the cells will begin to synthesize lots of T7 RNA polymerase. This T7 RNA polymerase can then bind to the T7 promoter sites on the plasmid and begin to synthesize RNA from both T7 RNA polymerase sites. Because the two single strands of RNA are complementary to each other they will form double stranded RNA within the bacterial cell. The IPTG induction allows us to "turn on" and express the plasmid gene of interest only when we want to and it allow us to make much higher levels of RNA for RNA interference than would be made without this induction.
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Another useful thing about E coli strain HT 115(DE3)is that this particular strain is '''deficient''' for the RNAaseIII enzyme that degrades double stranded RNA (dsRNA) in the bacterial cell. This allows for the accumulation of dsRNA in the cell and, thus, our ability to induce and RNAi effect! This ''E. coli'' strain carries a tetracyclin resistance gene so these cells can be selected on media containing tetracyclin, while the plasmid contains an ampicillin resistance gene that allows only transformed cells to grow on media containing ampicillin.
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Our goal is to upregulate production of dsRNA of our worm gene of interest in pL4440 vector plasmids containing that gene in HT115(DE3) bacteria.
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'''To induce your cultures, the lab specialist or your instructor:'''
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#Added 5 μL of 0.5 M IPTG to your culture. What is the effective concentration of IPTG in your culture?
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#Put your culture back in the 37°C incubator in the spinning wheel for approximately 3-4 hours.
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#While we have our journal article discussion in a more comfortable room, the bacteria will be working hard expressing T7 RNApolymerase and transcribing complementary mRNA of ''C. elegans' bli-1".
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'''To do after induction is complete:'''
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#Pour your culture into a 15 ml orange cap centrifuge tube.
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#Spin your culture in a table top centrifuge for 5 minutes at 3000 rpm.
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#Remove 3.5 ml of the supernatant.
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#Resuspend the bacterial pellet in the remaining 1.0 ml of supernatant - you are concentrating your bacteria.
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#In the laminar flow hood in L304 (the 220 lab) pipet a 200 μL aliquot of your induced bacteria onto the center of 4 '''feeding plates'''. Be careful not to tilt or jostle your plates so that the bacteria stay in a circle in the center of each plate. These plates contain the same NGM Lite medium used in our mapping series, except that they have been supplemented with 0.4 mM IPTG, 50 μg/mL ampicillin.
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#Allow the bacteria to be absorbed into the media before you move them. About 30-60 minutes.
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#Obtain 2 '''control''' plates - these plates contain the same NGM lite medium described above and the bacterial strain on them are identical to your RNAi feeder strain, ''except'' that the pL4440 plasmid is only expressing RNA from the vector - it lacks DNA specific to any worm genes.
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#Stack all 6 dry plates and put them in your lab section box with a labeled piece of your team color tape on top.
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#The plates will be stored at 4°C until you are ready to add worms.
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'''4 days before next lab:'''
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#Come into lab and find your stack of plates. For Friday, Monday and Tuesday sections your plates will be at room temperature by your worm picks. For Wednesday and Thursday sections your plates will be in the refrigerator in the lab - The first person to come in on Saturday or Sunday should remove the whole worm plate box and leave it on the counter near the worm picks so that the plates will be pre-warmed for your classmates. You should warm your own plates up to room temp before transferring your worms (hold them in your hands or put them in the 37C incubator by the door for a few minutes) or you will shock the worms with the cold medium.
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#On 2 of the experimental plates add 2 L4 wild type (N2) hermaphrodites
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#On 2 of the experimental plates add 2 L4 ''rrf-3'' hermaphrodites
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#On 1 of the control plates add 2 L4 wild type (N2) hermaphrodites
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#On 1 of the control plates add 2 L4 ''rrf-3'' hermaphrodites
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#Incubate the worms in your lab day box in the worm incubator set at 20°C.
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You will score your phenotypes in the next lab.
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== Assignment ==
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Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab.
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== Outline of Experimental Design for REVERSE Genetics Project ==
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'''Where are you now in this process?''' (What have you done so far? What's next?)
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'''A.''' Make the feeder strain of bacteria
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# Amplify gene of interest by PCR
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# Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation
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# Clean up DNA (remove enzymes)
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# Cloning: ligate gene into vector plasmid with amp resistance gene
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# Transform competent bacterial cells
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# Select for transformants on media with ampicillin
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# Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interest
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# Culture the selected colony from colony pcr to create a lot of copies of these bacteria
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# Isolate the cloned plasmid DNA from that cultured colony by miniprep
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# Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA
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# Select for transformants on media with ampicillin
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# Choose an isolated colony to culture and make lots of feeder strain bacteria
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# Induce expression of ''C. elegans'' gene dsRNA from the pL4440 vector in the bacteria by IPTG induction
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# Seed NM lite worm growth media plates with feeder strain produced as described
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'''B.''' Plate wild type ''C. elegans'' worms (N2 and ''rrf-3'' strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest).
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'''C.''' Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.
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'''Series3:'''
'''Series3:'''
[[BISC219/F12:RNA interference | RNA interference]]
[[BISC219/F12:RNA interference | RNA interference]]
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[[BISC219/F12: RNAi General Information |
RNAi General Information]]
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[[BISC219/F12: RNAi General Information |
Background Info on Project 3
]]
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[[BISC219/F12: Media Recipes | Media Recipes]]
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[[BISC219/F12: RNAi Lab 7 | Lab 7: ]]
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[[BISC219/F12: RNAi Lab 5 | Lab 5: Picking your gene to RNAi]]
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[[BISC219/F12: RNAi Lab 8 | Lab 8: ]]
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[[BISC219/F12: RNAi Lab 6 | Lab 6: Cloning your gene of interest
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[[BISC219/F12: RNAi Lab 9 | Lab 9: ]]
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[[BISC219/F12: RNAi Lab 7 | Lab 7:
Picking your transformant
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[[BISC219/F12: RNAi Lab 10 | Lab 10: ]]
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[[BISC219/F12: RNAi Lab 8 | Lab 8:
Plasmid purification and transformation
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[[BISC219/F12: RNAi Lab 9 | Lab 9:
Induction of bacteria for RNAi
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[[BISC219/F12: RNAi Lab 10 | Lab 10:
Scoring your worms
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[[BISC219/F12: RNAi Lab 11 | Lab 11: ]]
[[BISC219/F12: RNAi Lab 11 | Lab 11: ]]